P. Marx-Stoelting

Scientific principles for the identification of endocrine-disrupting chemicals: a consensus statement

Endocrine disruption is a specific form of toxicity, where natural and/or anthropogenic chemicals, known as “endocrine disruptors” (EDs), trigger adverse health effects by disrupting the endogenous hormone system. There is need to harmonize guidance on the regulation of EDs, but this has been hampered by what appeared as a lack of consensus among scientists. This publication provides summary information about a consensus reached by a group of world-leading scientists that can serve as the basis for the development of ED criteria in relevant EU legislation. Twenty-three international scientists from different disciplines discussed principles and open questions on ED identification as outlined in a draft consensus paper at an expert meeting hosted by the German Federal Institute for Risk Assessment (BfR) in Berlin, Germany on 11–12 April 2016. Participants reached a consensus regarding scientific principles for the identification of EDs. The paper discusses the consensus reached on background, definition of an ED and related concepts, sources of uncertainty, scientific principles important for ED identification, and research needs. It highlights the difficulty in retrospectively reconstructing ED exposure, insufficient range of validated test systems for EDs, and some issues impacting on the evaluation of the risk from EDs, such as non-monotonic dose–response and thresholds, modes of action, and exposure assessment. This report provides the consensus statement on EDs agreed among all participating scientists. The meeting facilitated a productive debate and reduced a number of differences in views. It is expected that the consensus reached will serve as an important basis for the development of regulatory ED criteria.

Combination Effects of (Tri)Azole Fungicides on Hormone Production and Xenobiotic Metabolism in a Human Placental Cell Line

Consumers are exposed to multiple residues of different pesticides via the diet. Therefore, EU legislation for pesticides requires the evaluation of single active substances as well as the consideration of combination effects. Hence the analysis of combined effects of substances in a broad dose range represents a key challenge to current experimental and regulatory toxicology. Here we report evidence for additive effects for (tri)azole fungicides, a widely used group of antifungal agents, in the human placental cell line Jeg-3. In addition to the triazoles cyproconazole, epoxiconazole, flusilazole and tebuconazole and the azole fungicide prochloraz also pesticides from other chemical classes assumed to act via different modes of action (i.e., the organophosphate chlorpyrifos and the triazinylsulfonylurea herbicide triflusulfuron-methyl) were investigated. Endpoints analysed include synthesis of steroid hormone production (progesterone and estradiol) and gene expression of steroidogenic and non-steroidogenic cytochrome-P-450 (CYP) enzymes. For the triazoles and prochloraz, a dose dependent inhibition of progesterone production was observed and additive effects could be confirmed for several combinations of these substances in vitro. The non-triazoles chlorpyrifos and triflusulfuron-methyl did not affect this endpoint and, in line with this finding, no additivity was observed when these substances were applied in mixtures with prochloraz. While prochloraz slightly increased aromatase expression and estradiol production and triflusulfuron-methyl decreased estradiol production, none of the other substances had effects on the expression levels of steroidogenic CYP-enzymes in Jeg-3 cells. For some triazoles, prochloraz and chlorpyrifos a significant induction of CYP1A1 mRNA expression and potential combination effects for this endpoint were observed. Inhibition of CYP1A1 mRNA induction by the AhR inhibitor CH223191 indicated AhR receptor dependence of this effect.

Hepatotoxic effects of (tri)azole fungicides in a broad dose range

Abstract The toxicological relevance of effects observed at molecular stage, which occur at dose levels well below classical no-observed adverse effect levels is currently subject to controversial scientific debate. While the importance of molecular effects for the identification of a mode of action or an adverse outcome pathway is undisputed, their impact for other regulatory purposes remains uncertain. Here, we report the results of a 28-day rat-feeding study including three widely used hepatotoxic (tri)azole fungicides (cyproconazole, epoxiconazole and prochloraz) administered individually at five dose levels, ranging from slightly above the reference values to a clear toxic effect dose. Parameters analysed included pathology, histopathology, clinical chemistry and particularly effects on the molecular level. Since azole fungicides are considered to cause liver toxicity by a mechanism involving the constitutive androstane receptor (CAR), a known CAR activator (phenobarbital, PB) was administered to investigate potential similarities between triazoles and PB-mediated liver toxicity by pathway-focused gene expression analysis. Our results show an increase in liver weights and additionally histopathological changes (hepatocellular hypertrophy) for all substances at the top dose levels. The effects on liver weight were most pronounced for cyproconazole by which also the animals receiving the next lower dose were affected. In addition, vacuolisation of hepatocytes was observed at the top dose level. No such findings were obtained with any substance at lower doses to which consumers and operators might be exposed to. In contrast, the expression of sensitive marker genes (like some cytochrome-P-450 isoforms) was significantly affected also at the lower dose levels. While some of these changes, like the induction of genes related to fatty acid and phospholipid metabolism (e.g. Fasn, Fat/Cd36, Ppargc1a) or xenobiotic metabolism (Cyp1a1, Cyp2b1, Cyp3a2), could be associated with high dose effects like hepatocellular vacuolisation or hypertrophy, a histopathological correlate was lacking for others.

Assessment strategies and decision criteria for pesticides with endocrine disrupting properties relevant to humans

There is growing concern that environmental substances with a potential to modulate the hormonal system may have harmful effects on human health. Consequently, a new EU regulation names endocrine disrupting properties as one of the cut-off criteria for the approval of plant protection products, although it currently fails to provide specific science-based measures for the assessment of substances with such properties. Since specific measures are to be presented by the European Commission in 2013 the development of assessment and decision criteria is a key challenge concerning the implementation of this new EU regulation. Proposals of such decision criteria for substances with potential endocrine disrupting properties in human health risk assessment were developed by the German Federal Institute for Risk Assessment (BfR) and discussed at an expert workshop in November 2009. Under consideration of the requirements laid down within the new plant protection product legislation and the scientific discussions during the workshop, a conceptual framework on evaluation of substances for endocrine disrupting properties in a regulatory context is presented in this paper. Central aspects of the framework include assessment of adversity of effects, establishment of a mode/mechanism of action in animals, considerations concerning the relevance of effects to humans and two options for a regulatory decision.

Application of omics data in regulatory toxicology: report of an international BfR expert workshop.

Abstract Advances in omics techniques and molecular toxicology are necessary to provide new perspectives for regulatory toxicology. By the application of modern molecular techniques, more mechanistic information should be gained to support standard toxicity studies and to contribute to a reduction and refinement of animal experiments required for certain regulatory purposes. The relevance and applicability of data obtained by omics methods to regulatory purposes such as grouping of chemicals, mode of action analysis or classification and labelling needs further improvement, defined validation and cautious expert judgment. Based on the results of an international expert workshop organized 2014 by the Federal Institute for Risk Assessment in Berlin, this paper is aimed to provide a critical overview of the regulatory relevance and reliability of omics methods, basic requirements on data quality and validation, as well as regulatory criteria to decide which effects observed by omics methods should be considered adverse or non-adverse. As a way forward, it was concluded that the inclusion of omics data can facilitate a more flexible approach for regulatory risk assessment and may help to reduce or refine animal testing.

Combination effects of azole fungicides in male rats in a broad dose range

Two 28-day feeding studies were performed in male rats to investigate combination effects of azole fungicides in a broad dose range. Following separate administration of cyproconazole, epoxiconazole, prochloraz, propiconazole, and tebuconazole at five dose levels, the first three compounds were selected to be administered in two different mixtures at three dose levels including very low doses. Here we present the data obtained by clinical observations, pathology, histopathology, clinical chemistry and haematology. The liver was the common main target organ of all compounds and their mixtures. In addition, epoxiconazole exhibited an effect on the adrenals. Furthermore, food consumption and efficiency and body weight (gain) were affected. Adverse effects of the combinations were observed at dose levels at which the individual substances caused similar effects. No evidence of adverse effects was found at dose levels below the previously established NOAELs. Our findings indicate that the concept of dose additivity appears sufficiently protective for risk assessment of the fungicides examined. Besides toxicological testing, tissue residues of the azole compounds in liver, testis and kidney were determined revealing remarkable differences following administration of the single substances and of the mixtures.

Relevance and reliability of experimental data in human health risk assessment of pesticides

ABSTRACT Evaluation of data relevance, reliability and contribution to uncertainty is crucial in regulatory health risk assessment if robust conclusions are to be drawn. Whether a specific study is used as key study, as additional information or not accepted depends in part on the criteria according to which its relevance and reliability are judged. In addition to GLP‐compliant regulatory studies following OECD Test Guidelines, data from peer‐reviewed scientific literature have to be evaluated in regulatory risk assessment of pesticide active substances. Publications should be taken into account if they are of acceptable relevance and reliability. Their contribution to the overall weight of evidence is influenced by factors including test organism, study design and statistical methods, as well as test item identification, documentation and reporting of results. Various reports make recommendations for improving the quality of risk assessments and different criteria catalogues have been published to support evaluation of data relevance and reliability. Their intention was to guide transparent decision making on the integration of the respective information into the regulatory process. This article describes an approach to assess the relevance and reliability of experimental data from guideline‐compliant studies as well as from non‐guideline studies published in the scientific literature in the specific context of uncertainty and risk assessment of pesticides. HIGHLIGHTSRelevant (appropriate) and reliable (trustworthy) data are needed for regulatory decision making.Selection of data used in risk assessment and weight of evidence can directly influence the decision making process.Transparent evaluation criteria are essential to understand why and how certain data are relied on.Specific requirements for experimental data from different sources for health risk assessment of pesticides are described.Adherence to good scientific and reporting practice is key to facilitate regulatory use of published research results.

Unexpected Effects of Propiconazole, Tebuconazole, and Their Mixture on the Receptors CAR and PXR in Human Liver Cells

Analyzing mixture toxicity requires an in-depth understanding of the mechanisms of action of its individual components. Substances with the same target organ, same toxic effect and same mode of action (MoA) are believed to cause additive effects, whereas substances with different MoAs are assumed to act independently. Here, we tested 2 triazole fungicides, propiconazole, and tebuconazole (Te), for individual and combined effects on liver toxicity-related endpoints. Both triazoles are proposed to belong to the same cumulative assessment group and are therefore thought to display similar and additive behavior. Our data show that Te is an antagonist of the constitutive androstane receptor (CAR) in rats and humans, while propiconazole is an agonist of this receptor. Both substances activate the pregnane X-receptor (PXR) and further induce mRNA expression of CYP3A4. CYP3A4 enzyme activity, however, is inhibited by propiconazole. For common targets of PXR and CAR, the activation of PXR by Te overrides CAR inhibition. In summary, propiconazole and Te affect different hepatotoxicity-relevant cellular targets and, depending on the individual endpoint analyzed, act via similar or dissimilar mechanisms. The use of molecular data based on research in human cell systems extends the picture to refine cumulative assessment group grouping and substantially contributes to the understanding of mixture effects of chemicals in biological systems.

Liver lobe and strain differences in the activity of murine cytochrome P450 enzymes

The cytochrome P450 (CYP) enzyme superfamily is the most important enzyme system for phase I biotransformation. For toxico- and pharmacokinetic studies, use of liver-based microsomes, including those of mice, is state-of-the-art to study CYP-dependent metabolism. However, reproducibility and interpretation of these data is still very variable, partly because current testing guidelines do not cover details on organ sampling and potential liver lobe differences. Hence, we analyzed CYP activity, CYP protein content, mRNA expression of CYP1A, CYP2C, CYP2D and CYP3A isozymes, and cytochrome P450 reductase (CPR) activity of the four different liver lobes and processus papillaris of male C57BL/6J mice in comparison to whole liver. Additionally, we used whole liver of Balb/cJ and 129S1/SvImJ for strain comparison. Our data show significant differences in CYP activity, being most prominent in lobus sinister lateralis and lobus medialis, and lowest in processus papillaris. These differences were not caused by varying Cyp gene expression or CYP protein level, but partly correspond with lobe specific CPR activities. We also observed significant strain differences in CYP mRNA expression and activities with overall high activities in 129S1/SvImJ mice and low activities in Balb/cJ mice compared to C57BL/6J mice. In addition, strain specific differences in CYP2C and CYP2D activity seem to be reflected in strain dependent differences in CPR activity. In summary, our results indicate that in mice CYP activity and gene expression are strain dependent and may vary highly between liver lobes. To ensure reproducibility and comparability of different probes and studies, this should be taken into account when liver samples are collected for the analysis of CYP-dependent metabolism.

Science-based decision matrix for the identification of endocrine disruptors for regulatory purposes

Recent EU legislation for chemical substances requires a particular assessment of endocrine disrupting properties that may cause adverse effects in humans. Especially for pesticidal active substances, measures concerning specific scientific criteria for the determination of endocrine disrupting properties should have been presented by the European Commission until December 2013. But presently, no specific science-based approach for the assessment of these substances has been agreed upon. This paper is discussing common scientific principles for the evaluation and grouping of substances with endocrine disrupting properties that may cause adverse effects in humans. A matrix-based approach is proposed to be applied in various fields of scientific evaluation of chemical substances, which is based on a scientific evaluation of all available data that may contribute to ensure a high level of protection of human health. This evaluation is expected to be proportionate, consistent and predictable to support administrative decisions in regulatory toxicology. However, a scientifically based categorisation in a decision matrix as a backbone for specific and legally binding rules should be performed according to the relevant EU regulations for the aforementioned groups of substances. Considering the complexity of the matter, it appears appropriate to base possible categorisation on considerations in a decision matrix, which take into account severity, reversibility, potency and consistency of an adverse effect. Based on this decision matrix it should be possible to allocate substances falling under the WHO/IPCS definition to categorise as endocrine disruptors (EDs) or even dispense such substances from categorisation.